Synergistic bacillus thuringiensis subsp. aizawai and bacillus thuringiensis subsp. kurstaki mixtures for diamondback moth, beet armyworm, sugarcane borer, soybean looper, corn earworm, cabbage looper, and southwestern corn borer control

ABSTRACT

The present invention generally relates to the use of synergistic amounts of  Bacillus thuringiensis  subsp.  aizawai  and  Bacillus thuringiensis  subsp.  kurstaki  for the control of diamondback moth, beet armyworm, sugarcane borer, soybean looper, corn earworm, cabbage looper and southwestern corn borer, wherein the ratio of  Bacillus thuringiensis  subsp.  kurstaki  to  Bacillus thuringiensis  subsp.  aizawai  is from about 1:0.001 to about 1:3.5.

FIELD OF THE INVENTION

The present invention generally relates to the use of synergisticamounts of Bacillus thuringiensis subsp. aizawai and Bacillusthuringiensis subsp. kurstaki for the control of diamondback moth, beetarmyworm, sugarcane borer, soybean looper, corn earworm, cabbage looperand southwestern corn borer.

BACKGROUND OF THE INVENTION

Lepidoptera is an order of insects which includes moths and butterflies.It is estimated that there are over 174,000 Lepidopteran species,included in an estimated 126 families. Lepidopteran species undergo acomplete metamorphosis during their life cycle. Adults mate and layeggs. The larvae that emerge from the eggs have a cylindrical body andchewing mouth parts. Larvae undergo several growth stages called instarsuntil they reach their terminal instar and then pupate. Lepidoptera thenemerge as adult butterflies or moths.

While some Lepidoptera species are generally considered beneficialorganisms due to their aesthetic appeal, many species cause devastatingdamage to crops. Specifically, diamondback moths, beet armyworms,sugarcane borers, soybean loopers, corn earworm, cabbage looper andsouthwestern corn borer are especially problematic to crop growers.

Diamondback moths (Plutella xylostella) are a widespread pest that candisperse long distances. Diamondback moth larvae eat the leaves, buds,flowers and seed-buds of cruciferous plants. A heavy infestation cancompletely remove all foliar tissue from a plant leaving only the leafveins. Even a lighter infestation can result in the unsuitability of anentire lot of produce for sale. In the past, diamondback moths have beentreated with a variety of insecticides including pyrethroids.

Beet armyworms (Spodoptera exigua) are another widespread pest that isdifficult to control. The larvae are voracious eaters that defoliatehost plants. Older instars can also burrow into the plants. The damageto the host plant renders it unmarketable. Beet armyworms are pests onnumerous types of crops.

Sugarcane borers (Diatraea saccharalis) mostly attack sugarcane andsweet corn crops, but will also infest other host plants. The larvaeburrow into the stalks of the older plants causing the plant to weakenand break off or die. In younger plants, the inner whorl of leaves willdie and yields will be impacted. Secondary fungal infections may alsocommonly occur as a result of seed cane predation. There has been somesuccess in controlling sugarcane borers with insecticides but they needto be applied to the plants before the larvae burrow into the stalks.

Soybean loopers (Chrysodeixis includens) are a moth that is prevalent inNorth and South America. The larvae of soybean loopers can inflict heavyfoliage damage resulting in significant crop loss. Soybean loopers aredifficult to control with insecticides. Infestation of soybean looperscan be exacerbated after a non-selective insecticide removes the soybeanloopers' natural predators.

Corn earworms (Helicoverpa zea) have been referred to as the most costlycrop pest in the United States. Corn earworms are difficult to controlwith insecticides because they can burrow into the plants and avoidexposure to insecticide applications. Corn earworms have numerousnatural predators but predators and parasitoids alone are not effectiveat preventing crop plant damage by Helicoverpa zea.

Cabbage loopers (Trichoplusia ni) are a destructive crop pest in NorthAmerica. During their larval stage, they eat three-times their bodyweight in plant material a day. Once they are established in a cropfield, they are difficult to control.

Southwestern corn borers (Diatraea grandiosella) cause crop damage inthe United States and Mexico. It is estimated that southwestern cornborers cause millions of dollars of damage each year. The first larvaethat emerge after overwintering feed on the whorl of the plant and cancause total destruction of the plant (dead heart). The second generationthat emerges later in the growing season feeds on leaf axils andeventually bores into the stalks which can cause girdling and can alsolead to death of the plant.

Bacillus thuringiensis is a natural soil bacterium. Many Bacillusthuringiensis strains produce crystal proteins during sporulation calledδ-endotoxins which can be used as biological insecticides. Bacillusthuringiensis, subspecies aizawai and kurstaki, produce crystals whichparalyze the digestive system of some larvae within minutes. The larvaeeventually die off from the multiple deleterious effects from toxininteractions with the gut tissues of the target pest. Bacillusthuringiensis subsp. aizawai is commercially available as XenTari®(available from Valent BioSciences Corporation, XenTari is a registeredtrademark of Valent BioSciences Corporation). Bacillus thuringiensissubsp. kurstaki is commercially available as Dipel® (available fromValent BioSciences Corporation, Dipel is a registered trademark ofValent BioSciences Corporation). Sympatico™ is a commerical product thatis a combination of Bacillus thuringiensis subsp. aizawai and Bacillusthuringiensis subsp. kurstaki fermentation solids, spores, and toxins(also available from Valent BioSciences Corporation).

One advantage of using Bacillus thuringiensis subsp. aizawai andBacillus thuringiensis subsp. kurstaki is that they are target specific.They do not harm humans or other non-target species. Frequently whenplants are treated with a non-selective insecticide, the insecticidealso kills natural predators of other pests. This can cause a reboundeffect in the target insect or other opportunistic pest species. Forexample, after applying a non-selective pesticide to kill corn borers, aspider mite infestation might occur because the non-selective pesticidealso killed the spider mites' natural predators.

Yet another advantage of Bacillus thuringiensis subsp. aizawai andBacillus thuringiensis subsp. kurstaki is that they can be used onorganic crops. With no mandated pre-harvest interval, it can also beused on crops right before harvest. This provides organic growers, whohave few options for pest control, a safe and effective way to manageinsect infestations that could ultimately ruin an entire crop.

Accordingly, there is a need for safe and effective ways to controldiamondback moth, beet armyworm, sugarcane borer, soybean looper, cornearworm, cabbage looper and southwestern corn borer. These methodsshould be easy to apply, have increased efficacy, and be cost effective.

SUMMARY OF THE INVENTION

The present invention is directed to methods for controlling diamondbackmoth (Plutella xylostella), beet armyworm (Spodoptera exigua), sugarcaneborer (Diatraea saccharalis), soybean looper (Chrysodeixis includens),corn earworm (Helicoverpa zea), cabbage looper (Trichoplusia ni), andsouthwestern corn borer (Diatraea grandiosella) comprising applying asynergistic amount of Bacillus thuringiensis subsp. aizawai and Bacillusthuringiensis subsp. kurstaki to a plant, wherein the ratio of Bacillusthuringiensis subsp. kurstaki to Bacillus thuringiensis subsp. aizawaiis from about 1:0.001 to about 1:3.5.

DETAILED DESCRIPTION OF THE INVENTION

Applicant discovered that the use of Bacillus thuringiensis subsp.kurstaki and Bacillus thuringiensis subsp. aizawai at a ratio range offrom about 1:0.001 to about 1:3.5 provided unexpected synergisticeffects against specific Lepidopteran species. This synergy wasunexpected because the response to the treatment was highly speciesspecific and unpredictable.

The Bacillus thuringiensis subsp. aizawai and Bacillus thuringiensissubsp. kurstaki synergistic mixtures are safe to use on edible plants.The components of the mixtures are safe to use on organically grownedible plants as well. Further, the components of the mixtures aretarget specific and pose low to no risk to beneficial insects oranimals.

Another advantage of the present invention is that the combination ofBacillus thuringiensis subsp. aizawai and Bacillus thuringiensis subsp.kurstaki aligns with Integrated Pest Management (IPM) principles. Thepresent mixture two species of Bacillus thuringiensis exposes the larvaeto a broader range of toxins. This means that the mixture of Bacillusthuringiensis subsp. aizawa and Bacillus thuringiensis subsp. kurstakican be applied repeatedly in the same season and year after year withminimal risk of resistance developing.

Yet another advantage of the present invention is that it allows forless Bacillus thuringiensis to be applied to the plant. For example,within label rates, sub-lethal doses of each can be applied to achieve alethal dose and control of the larvae. This allows for a significantcost saving to the grower.

A further advantage is that Bacillus thuringiensis subsp. aizawai andBacillus thuringiensis subsp. kurstaki are target-specific. This meansthat humans and other, non-target organisms—such as natural predators ofdiamondback moth, beet armyworm, sugarcane borer, soybean looper, cornearworm, cabbage looper, and southwestern corn borer—will not be harmedby the methods of the present invention.

In an embodiment, the present invention is directed to methods forcontrolling a crop plant pest selected from the group consisting ofdiamondback moth (Plutella xylostella), beet armyworm (Spodopteraexigua), sugarcane borer (Diatraea saccharalis), soybean looper(Chrysodeixis includens), corn earworm (Helicoverpa zea), cabbage looper(Trichoplusia ni), and southwestern corn borer (Diatraea grandiosella)comprising applying a synergistic amount of Bacillus thuringiensissubsp. aizawai and Bacillus thuringiensis subsp. kurstaki to a plant,wherein the ratio of Bacillus thuringiensis subsp. kurstaki to Bacillusthuringiensis subsp. aizawai is from about 1:0.001 to about 1:3.5.

As used herein, “crop plant pest” only refers to diamondback moth(Plutella xylostella), beet armyworm (Spodoptera exigua), sugarcaneborer (Diatraea saccharalis), soybean looper (Chrysodeixis includens),corn earworm (Helicoverpa zea), cabbage looper (Trichoplusia ni), andsouthwestern corn borer (Diatraea grandiosella).

In a preferred embodiment, the ratio of Bacillus thuringiensis subsp.kurstaki to Bacillus thuringiensis subsp. aizawai is from about 1:0.01to about 1:3. In a more preferred embodiment, the ratio of Bacillusthuringiensis subsp. kurstaki to Bacillus thuringiensis subsp. aizawaiis from about 1:0.1 to about 1:2.5.

In another embodiment, the present invention is directed to methods forcontrolling a crop plant pest wherein the amount of Bacillusthuringiensis subsp. aizawai/Bacillus thuringiensis subsp. kurstaki isfrom about 5 to about 400 grams per hectare. In a preferred embodiment,the amount of Bacillus thuringiensis subsp. aizawai/Bacillusthuringiensis subsp. kurstaki is from about 10 to about 350 grams perhectare. In a more preferred embodiment, the amount of Bacillusthuringiensis subsp. aizawai/Bacillus thuringiensis subsp. kurstaki isfrom about 25 to about 300 grams per hectare.

In a further embodiment, the present invention is directed to methodsfor controlling a crop plant pest wherein the amount of Bacillusthuringiensis subsp. aizawai/Bacillus thuringiensis subsp. kurstaki isfrom about 7,000 to about 200,000 IU/mg. In a preferred embodiment, theamount of Bacillus thuringiensis subsp. aizawai/Bacillus thuringiensissubsp. kurstaki is from about 20,000 to about 170,000 IU/mg. In a morepreferred embodiment, the amount of Bacillus thuringiensis subsp.aizawai/Bacillus thuringiensis subsp. kurstaki is from about 25,000 toabout 100,000 IU/mg.

In yet another embodiment, the present invention is directed to methodsfor controlling a crop plant pest wherein the total amount of Bacillusthuringiensis is from about 1,000 to about 100,000 Spodoptera U/mg. In apreferred embodiment, the amount of Bacillus thuringiensis is from about10,000 to about 90,000 Spodoptera U/mg. In a more preferred embodiment,the amount of Bacillus thuringiensis is from about 15,000 to about70,000 Spodoptera U/mg.

Although in some embodiments, the rates of Bacillus thuringiensis subsp.aizawai and Bacillus thuringiensis subsp. kurstaki are expressed ingrams/hectare, IU/mg, or Spodoptera U/mg, the invention is not limitedto these methods of measuring potency. If other products are developedor marketed with other potency measurements, it is within the knowledgeof one of skill in the art, based on Applicant's teaching herein, toconvert the rates to effective amounts consistent with the inventionherein to achieve synergistic control of the target crop plant pest.

Further, the present invention is not limited to a specific type offormulation. For example, in the examples herein, unformulated technicalpowders were used as the source of Bacillus thuringiensis subsp. aizawaiand Bacillus thuringiensis kurstaki. However, formulations may be usedas the source of these actives, including but not limited to,emulsifiable suspensions, wettable powder formulations, waterdispersible granules, dry flowable granules, and other granules.

Suitable Bacillus thuringiensis subsp. aizawai subspecies strainsinclude, but are not limited to, VBTS-1857, GB413 and GC-91, andtransconjugated, recombinant and/or genetically engineered subspeciesthereof.

Suitable Bacillus thuringiensis subsp. aizawai commercial productsinclude, but are not limited to, XenTari® (as indicated above, availablefrom Valent BioSciences Corporation, XenTari is a registered trademarkof Valent BioSciences Corporation), Solbit (available from Green BiotechCompany), Bacchus® (available from Certis, Bacchus is registeredtrademark of Certis USA, L.L.C.), Agree® (available from Certis, Agreeis registered trademark of Certis USA, L.L.C.), Jackpot® (available fromCertis, Jackpot is registered trademark of Certis USA, L.L.C.), andTurex (available from Certis, Turex is registered trademark of CertisUSA, L.L.C.).

Suitable Bacillus thuringiensis subsp. kurstaki subspecies strainsinclude, but are not limited to, VBTS-2546, VBTS-2528, BMP-123, EG-2348,EVB113-19, HD-1, PB-54, SA-11, SA-12, SB4, Z-52, EG-7841, andtransconjugated, recombinant and/or genetically engineered subspeciesthereof.

Suitable Bacillus thuringiensis subsp. kurstaki commercial productsinclude, but are not limited to, DiPel® (as indicated above, availablefrom Valent BioSciences Corporation, DiPel is a registered trademark ofValent BioSciences Corporation), BMP 123 (available from BeckerMicrobials), Lepinox Plus (available from CBC Biogard), Rapax (availablefrom CBC Biogard), Bioprotec 3P (available from AEF Global), BacillusChemia (available from Chemia), Biolary (available from Agrimix),Bacillus Agrogen WP (available from Yaser Ltd), Merger/Belthirul(available from Probelte), Delfin® (available from Certis, Delfin is aregistered trademark of Certis USA, L.L.C.), Javelin® WG (available fromCertis, Javelin is a registered trademark of Certis USA, L.L.C.),Costar® (available from Certis, Costar is a registered trademark ofCertis USA, L.L.C.), Deliver® (available from Certis, Deliver is aregistered trademark of Certis USA, L.L.C.), BeTa Pro (available fromBASF), Biolep (available from Biotech International Ltd), Full-Bac WDG(available from Becker Microbial), Bacillus MiPeru WP (available fromManejos Integrados Peru SA), and Crymax® (available from Certis, Crymaxis a registered trademark of Certis USA, L.L.C.).

In a further embodiment, the present invention is directed to methodsfor controlling a crop plant pest comprising applying a synergisticamount of Bacillus thuringiensis subsp. aizawai and Bacillusthuringiensis subsp. kurstaki wherein the ratio of Bacillusthuringiensis subsp. aizawai to Bacillus thuringiensis subsp. kurstakiis from about 1:0.001 to about 1:3.5, and wherein the plant is selectedfrom the group consisting of root and tuber vegetables, bulb vegetables,leafy non-brassica vegetables, leafy brassica vegetables, succulent ordried legumes, fruiting vegetables, cucurbit vegetables, citrus fruits,pome fruits, stone fruits, berry and small fruits, tree nuts, cerealgrains, forage and fodder grasses and hay, non-grass animal feeds,herbs, spices, flowers, bedding plants, ornamental flowers, artichoke,asparagus, coffee, cotton, tropical fruits, hops, malanga, peanut,pomegranate, oil seed vegetables, sugarcane, tobacco, turf, andwatercress.

In another embodiment, the crop plant is genetically modified. A“genetically modified” crop plant is one that has had specific genesremoved, modified or additional gene copies of native or foreign DNA.The change in the crop plant's DNA may result in can result in changesin the type or amount of RNA, proteins and/or other molecules that thecrop plant produces which may affect its response to abiotic (e.g.herbicide) or biotic (e.g. insects) stresses, and/or affect its growth,development, or yield.

In a preferred embodiment, the root and tuber vegetables are selectedfrom the group consisting of arracacha, arrowroot, Chinese artichoke,Jerusalem artichoke, garden beet, sugar beet, edible burdock, ediblecanna, carrot, bitter cassava, sweet cassava, celeriac, root chayote,turnip-rooted chervil, chicory, chufa, dasheen (taro), ginger, ginseng,horseradish, leren, turnip-rooted parsley, parsnip, potato, radish,oriental radish, rutabaga, salsify, black salsify, Spanish salsify,skirret, sweet potato, tanier, turmeric, turnip, yam bean, true yam, andcultivars, varieties and hybrids thereof.

In another preferred embodiment, the bulb vegetables are selected fromthe group consisting of fresh chive leaves, fresh Chinese chive leaves,bulb daylily, elegans Hosta, bulb fritillaria, fritillaria leaves, bulbgarlic, great-headed bulb garlic, serpent bulb garlic, kurrat, lady'sleek, leek, wild leek, bulb lily, Beltsville bunching onion, bulb onion,Chinese bulb onion, fresh onion, green onion, macrostem onion, pearlonion, potato bulb onion, potato bulb, tree onion tops, Welsh oniontops, bulb shallot, fresh shallot leaves, and cultivars, varieties andhybrids thereof.

In a further embodiment, the leafy non-brassica vegetables are selectedfrom the group consisting of Chinese spinach Amaranth, leafy Amaranth,arugula (roquette), cardoon, celery, Chinese celery, celtuce, chervil,edible-leaved chrysanthemum, garland chrysanthemum, corn salad, gardencress, upland cress, dandelion, dandelion leaves, sorrels (dock), endive(escarole), Florence fennel, head lettuce, leaf lettuce, orach, parsley,garden purslane, winter purslane, radicchio (red chicory), rhubarb,spinach, New Zealand spinach, vine spinach, Swiss chard, Tampala, andcultivars, varieties and hybrids thereof.

In another embodiment, the leafy brassica vegetables are selected fromthe group consisting of broccoli, Chinese broccoli (gai lon), broccoliraab (rapini), Brussels sprouts, cabbage, Chinese cabbage (bok choy),Chinese napa cabbage, Chinese mustard cabbage (gai choy), cauliflower,cavalo broccoli, collards, kale, kohlrabi, mizuna, mustard greens,mustard spinach, rape greens, and cultivars, varieties and hybridsthereof.

In yet another embodiment, the succulent or dried vegetable legumes areselected from the group consisting of Lupinus beans, Phaseolus beans,Vigna beans, broad beans (fava), chickpea (garbanzo), guar, jackbean,lablab bean, lentil, Pisum peas, pigeon pea, soybean, immature seedsoybean, sword bean, peanut, and cultivars, varieties and hybridsthereof. In a preferred embodiment, the Lupinus beans include grainlupin, sweet lupin, white lupin, white sweet lupin, and hybrids thereof.In another preferred embodiment, the Phaseolus beans include field bean,kidney bean, lima bean, navy bean, pinto bean, runner bean, snap bean,tepary bean, wax bean, and hybrids thereof. In yet another preferredembodiment, the Vigna beans include adzuki bean, asparagus bean,blackeyed bean, catjang, Chinese longbean, cowpea, Crowder pea, mothbean, mung bean, rice bean, southern pea, urd bean, yardlong bean, andhybrids thereof. In another embodiment, the Pisum peas include dwarfpea, edible-podded pea, English pea, field pea, garden pea, green pea,snow pea, sugar snap pea, and hybrids thereof. In a preferredembodiment, the dried vegetable legume is soybean. In a more preferredembodiment, the dried vegetable legume is genetically modified soybean.

In a further embodiment, the fruiting vegetables are selected from thegroup consisting of bush tomato, cocona, currant tomato, gardenhuckleberry, goji berry, groundcherry, martynia, naranjilla, okra, peaeggplant, pepino, peppers, non-bell peppers, roselle, Scout tomatofields roselle, eggplant, scarlet eggplant, African eggplant, sunberry,tomatillo, tomato, tree tomato, and cultivars, varieties and hybridsthereof. In a preferred embodiment, the peppers include bell peppers,chili pepper, cooking pepper, pimento, sweet peppers, and hybridsthereof.

In an embodiment, the cucurbit vegetables are selected from the groupconsisting of Chayote, Chayote fruit, waxgourd (Chinese preservingmelon), citron melon, cucumber, gherkin, edible gourds, Momordicaspecies, muskmelons, pumpkins, summer squashes, winter squashes,watermelon, and cultivars, varieties and hybrids thereof. In a preferredembodiment, edible gourds include hyotan, cucuzza, hechima, Chineseokra, and hybrids thereof. In another preferred embodiment, theMomordica vegetables include balsam apple, balsam pear, bittermelon,Chinese cucumber, and hybrids thereof. In another preferred embodiment,the muskmelon include true cantaloupe, cantaloupe, casaba, crenshawmelon, golden pershaw melon, honeydew melon, honey balls, mango melon,Persian melon, pineapple melon, Santa Claus melon, snake melon, andhybrids thereof. In yet another preferred embodiment, the summer squashinclude crookneck squash, scallop squash, straightneck squash, vegetablemarrow, zucchini, and hybrids thereof. In a further preferredembodiment, the winter squash includes butternut squash, calabaza,hubbard squash, acorn squash, spaghetti squash, and hybrids thereof.

In another embodiment, the citrus fruits are selected from the groupconsisting of limes, calamondin, citron, grapefruit, Japanese summergrapefruit, kumquat, lemons, Mediterranean mandarin, sour orange, sweetorange, pummel, Satsuma mandarin, tachibana orange, tangelo, mandarintangerine, tangor, trifoliate orange, uniq fruit, and cultivars,varieties and hybrids thereof. In a preferred embodiment, the limes areselected from the group consisting of Australian desert lime, Australianfinger lime, Australian round lime, Brown River finger lime, mount whitelime, New Guinea wild lime, sweet lime, Russell River lime, Tahiti lime,and hybrids thereof.

In an embodiment, the pome fruits are selected from the group consistingof apple, azarole, crabapple, loquat, mayhaw, medlar, pear, Asian pear,quince, Chinese quince, Japanese quince, tejocote, and cultivars,varieties and hybrids thereof.

In another embodiment, the stone fruits are selected from the groupconsisting of apricot, sweet cherry, tart cherry, nectarine, peach,plum, Chicksaw plum, Damson plum, Japanese plum, plumcot, fresh prune,and cultivars, varieties and hybrids thereof.

In a further embodiment, the berries and small fruits are selected fromthe group consisting of Amur river grape, aronia berry, bayberry,bearberry, bilberry, blackberry, blueberry, lowbush blueberry, highbushblueberry, buffalo currant, buffaloberry, che, Chilean guava,chokecherry, chokeberry, cloudberry, cranberry, highbush cranberry,black currant, red currant, elderberry, European barberry, gooseberry,grape, edible honeysuckle, huckleberry, jostaberry, Juneberry (Saskatoonberry), lingonberry, maypop, mountain pepper berries, mulberry,muntries, native currant, partridgeberry, phalsa, pincherry, blackraspberry, red raspberry, riberry, salal, schisandra berry, seabuckthorn, serviceberry, strawberry, wild raspberry, and cultivars,varieties and hybrids thereof. In a preferred embodiment, theblackberries include Andean blackberry, arctic blackberry, bingleberry,black satin berry, boysenberry, brombeere, California blackberry,Chesterberry, Cherokee blackberry, Cheyenne blackberry, commonblackberry, coryberry, darrowberry, dewberry, Dirksen thornless berry,evergreen blackberry, Himal ay ab erry, hullberry, lavacaberry,loganberry, lowberry, Lucreliaberry, mammoth blackberry, marionberry,mora, mures deronce, nectarberry, Northern dewberry, olallieberry,Oregon evergreen berry, phenomenalberry, rangeberry, ravenberry,rossberry, Shawnee blackberry, Southern dewberry, tayberry, youngberry,zarzamora, and hybrids thereof.

In another embodiment, the tree nuts are selected from the groupconsisting of almond, beech nut, Brazil nut, butternut, cashew,chestnut, chinquapin, hazelnut (filbert), hickory nut, macadamia nut,pecan, pistachio, black walnut, English walnut, and cultivars, varietiesand hybrids thereof.

In a further embodiment, the cereal grains are selected from the groupconsisting of barley, buckwheat, pearl millet, proso millet, oats, corn,field corn, sweet corn, seed corn, popcorn, rice, rye, sorghum (milo),sorghum species, grain sorghum, sudangrass (seed), teosinte, triticale,wheat, wild rice, and cultivars, varieties and hybrids thereof. In apreferred embodiment, the cereal grain is corn. In a more preferredembodiment, the cereal grain is genetically modified corn.

In yet another embodiment, the grass forage, fodder and hay are selectedfrom the group consisting of grasses that are members of the Gramineaefamily except sugarcane and those species included in the cereal grainsgroup, pasture and range grasses, and grasses grown for hay or silage.In further embodiments, the Gramineae grasses may be green or cured.

In an embodiment, the non-grass animal feeds are selected from the groupconsisting of alfalfa, velvet bean, trifolium clover, melilotus clover,kudzu, lespedeza, lupin, sainfoin, trefoil, vetch, crown vetch, milkvetch, and cultivars, varieties and hybrids thereof.

In another embodiment, the herbs and spices are selected from the groupconsisting of allspice, angelica, anise, anise seed, star anise, annattoseed, balm, basil, borage, burnet, chamomile, caper buds, caraway, blackcaraway, cardamom, cassia bark, cassia buds, catnip, celery seed,chervil, chive, Chinese chive, cinnamon, clary, clove buds, corianderleaf, coriander seed, costmary, culantro leaves, culantro seed, cilantroleaves, cilantro seed, cumin, dillweed, dill seed, fennel, commonfennel, Florence fennel seed, fenugreek, grains of paradise, horehound,hyssop, juniper berry, lavender, lemongrass, leaf lovage, seed lovage,mace, marigold, marjoram, mint, mustard seed, nasturtium, nutmeg,parsley, pennyroyal, black pepper, white pepper, poppy seed, rosemary,rue, saffron, sage, summer savory, winter savory, sweet bay, tansy,tarragon, thyme, vanilla, wintergreen, woodruff, wormwood, andcultivars, varieties and hybrids thereof. In a preferred embodiment, themints are selected from the group consisting of spearmint, peppermint,and hybrids thereof.

In yet another embodiment, artichokes are selected from the groupconsisting of Chinese artichoke, Jerusalem artichoke, and cultivars,varieties and hybrids thereof.

In an embodiment, the tropical fruits are selected from the groupconsisting of anonna, avocado, fuzzy kiwifruit, hardy kiwifruit, banana,plantain, caimito, carambola (star fruit), guava, longan, sapodilla,papaya, passion fruit, mango, lychee, jackfruit, dragon fruit, mameysapote, coconut cherimoya, canistrel, monster, wax jambu, pomegranate,rambutan, pulasan, Pakistani mulberry, langsat, chempedak, durian, figpineapple, jaboticaba, mountain apples, pineapple, and cultivars,varieties and hybrids thereof.

In a further embodiment, the oil seed vegetables are selected from thegroup consisting of borage, calendula, castor oil plant, tallowtree,cottonseed, crambe, cuphea, echium, euphorbia, evening primrose, flaxseed, gold of pleasure, hare's ear, mustard, jojoba, lesquerella,lunaria, meadowfoam, milkweed, niger seed, oil radish, poppy seed,rosehip, sesame, stokes aster, sweet rocket, tallowwood, tea oil plant,vermonia, canola, or oil rapeseed, safflower, sunflower, and cultivars,varieties and hybrids thereof.

The synergistic amounts of Bacillus thuringiensis subsp. aizawai andBacillus thuringiensis subsp. kurstaki may be applied to seeds, foliage,or an area where a plant is intended to grow.

The synergistic amounts of Bacillus thuringiensis subsp. aizawai andBacillus thuringiensis subsp. kurstaki may be applied once or many timesduring a growing season. If Bacillus thuringiensis subsp. aizawai andBacillus thuringiensis subsp. kurstaki are applied more than one time,the total amount applied should not exceed a yearly maximum rate asdetermined by environmental protection agencies or relevant label rates.

As used herein, “plant” refers to at least one plant and not a plantpopulation.

As used herein, “control” or “controlling” means a decline in the amountof damage to the plants from the larvae, reduction of pest population,interference with life cycle development or other physiological orbehavioral effect that results in plant protection.

As used herein, all numerical values relating to amounts, weightpercentages and the like, are defined as “about” or “approximately” eachparticular value, plus or minus 10%. For example, the phrase “at least5.0% by weight” is to be understood as “at least 4.5% to 5.5% byweight.” Therefore, amounts within 10% of the claimed values areencompassed by the scope of the claims.

The disclosed embodiments are simply exemplary embodiments of theinventive concepts disclosed herein and should not be considered aslimiting, unless so stated.

The following examples are intended to illustrate the present inventionand to teach one of ordinary skill in the art how to make and use theinvention. They are not intended to be limiting in any way.

EXAMPLES

The following examples illustrate the synergy of Bacillus thuringiensissubsp. aizawai (“Bta”) and Bacillus thuringiensis subsp. kurstaki(“Btk”) when controlling diamondback moth, beet armyworm, sugarcaneborer, soybean looper, corn earworm, cabbage looper, and southwesterncorn borer. Technical grade powders were used in the following examples.In each example below, the studies were conducted as follows.

For these tests, standardized laboratory leaf dip methods were used toinoculate plant material with treatment(s). Dry, treated leaves wereplaced into Petri dishes (100×25 mm) containing filter paper wetted with500 μl of distilled H₂O (“dH₂O”). Each dish was then infested withbetween 5 and 10 larvae, dependent on species. Efficacy ratings weretaken at specified intervals. Synergy ratings were calculated for eachtest.

Example 1 Diamondback Moth

In this study, the response of diamondback moth larvae to synergisticamounts of Bacillus thuringiensis subsp. aizawai (“Bta”) and Bacillusthuringiensis subsp. kurstaki (“Btk”) was observed. The results of thisstudy can be seen below in Table 1.

TABLE 1 % Efficacy Time after Neg. treatment Control Btk + Bta Synergy(h) dH₂O Bta Btk (Ratio 1:3.2) Ratio 24 0 3 3 6 1.02 48 0 7 20 52 2.02

As seen in Table 1, the mixtures of the present invention provided amore than additive effect. By using the following formula, Applicant wasable to determine that this response was synergistic: %C_(exp)=A+B−(AB/100).

% C_(exp)=A+B−(AB/100), where % C_(exp) is the expected efficacy and “inwhich A and B are the control levels given by the single [insecticides].If the ratio between the experimentally observed efficacy of the mixtureC_(obs) and the expected efficacy of the mixture is greater than 1,synergistic interactions are present in the mixture.” (Gisi,Synergisitic Interaction of Fungicides in Mixtures, The AmericanPhytopathological Society, 86:11, 1273-1279, 1996). Adopting aconservative approach, Applicant will determine if synergy is present atratios of ≧1.15.

Bacillus thuringiensis subsp. aizawai was applied at a concentration of10 ppm (10 μg/ml). Bacillus thuringiensis subsp. kurstaki was applied ata concentration of 3.125 ppm (3.125 μg/ml). The Bacillus thuringiensissubsp. aizawai/Bacillus thuringiensis subsp. kurstaki mixture wasapplied at a concentration of 10 ppm Bacillus thuringiensis subsp.aizawai and 3.125 ppm Bacillus thuringiensis subsp. kurstaki.

A synergy ratio that is indicative of synergy is this assay is apredictor of the synergy that will be seen in the field at normal fieldrates (or at rates that occur naturally as the active ingredients aredegraded over time by exposure to rain, UV radiation, and temperatureextremes). This assay was chosen for its ability to accurately predictmortality rates of larvae in the field.

The results of this calculation indicated that the synergy ratio was1.02 at 24 hours and 2.02 at 48 hours. As a finding of higher than 1 isindicative of synergy (per Gisi, or even ≧1.15 per Applicant), the ratioof 2.02 is synergistic. Synergy was shown at a ratio of Bacillusthuringiensis subsp. kurstaki to Bacillus thuringiensis subsp. aizawaiof 1:3.2.

Example 2 Beet Armyworm

In this study, the response of beet armyworm larvae to synergisticamounts of Bacillus thuringiensis subsp. aizawai and Bacillusthuringiensis subsp. kurstaki was observed. The results of this studycan be seen below in Table 2.

TABLE 2 % Efficacy Time after Neg. treatment Control Btk + Bta Synergy(h) dH₂O Bta Btk (Ratio 1:3.2) Ratio 24 0 0 7 10 1.43 48 0 0 7 17 2.43

As seen in Table 2, the mixtures of the present invention provided amore than additive effect. By using the following formula, Applicant wasable to determine that this response was synergistic: %C_(exp)=A+B−(AB/100).

Bacillus thuringiensis subsp. aizawai was applied at a concentration of10 ppm (10 μg/ml). Bacillus thuringiensis subsp. kurstaki was applied ata concentration of 3.125 ppm (3.125 μg/ml). The Bacillus thuringiensissubsp. aizawai/Bacillus thuringiensis subsp. kurstaki mixture wasapplied at a concentration of 10 ppm Bacillus thuringiensis subsp.aizawai and 3.125 ppm Bacillus thuringiensis subsp. kurstaki.

The results of this calculation indicated that the synergy ratio was1.43 at 24 hours and 2.43 at 48 hours. As a finding of higher than 1 isindicative of synergy, the ratios of 1.43 and above are synergistic.Synergy was shown at a ratio of Bacillus thuringiensis subsp. kurstakito Bacillus thuringiensis subsp. aizawai of 1:3.2.

Example 3 Cabbage Looper

In this study, the response of cabbage looper larvae to synergisticamounts of Bacillus thuringiensis subsp. aizawai and Bacillusthuringiensis subsp. kurstaki was observed. The results of this studycan be seen below in Table 3.

TABLE 3 % Efficacy Time after Neg. treatment Control Btk + Bta Synergy(h) dH₂O Bta Btk (Ratio 1:3.2) Ratio 24 0 0 0 3 n/a 48 0 3 3 16 2.71

As seen in Table 3, the mixtures of the present invention provided amore than additive effect. By using the following formula, Applicant wasable to determine that this response was synergistic: %C_(exp)=A+B−(AB/100).

Bacillus thuringiensis subsp. aizawai was applied at a concentration of10 ppm (10 μg/ml). Bacillus thuringiensis subsp. kurstaki was applied ata concentration of 3.125 ppm (3.125 μg/ml). The Bacillus thuringiensissubsp. aizawai/Bacillus thuringiensis subsp. kurstaki mixture wasapplied at a concentration of 10 ppm Bacillus thuringiensis subsp.aizawai and 3.125 ppm Bacillus thuringiensis subsp. kurstaki.

The results of this calculation indicated that the synergy ratio was2.71 at 48 hours. As a finding of higher than 1 is indicative ofsynergy, the ratio of 2.71 is clearly synergistic. Synergy was shown ata ratio of Bacillus thuringiensis subsp. kurstaki to Bacillusthuringiensis subsp. aizawai of 1:3.2.

Example 4 Sugarcane Borer

In this study, the response of sugarcane borer larvae to synergisticamounts of Bacillus thuringiensis subsp. aizawai and Bacillusthuringiensis subsp. kurstaki was observed. The results of this studycan be seen below in Table 4.

TABLE 4 % Efficacy Time after Neg. treatment Control Btk + Bta Synergy(h) dH₂O Bta Btk (Ratio 1:3.2) Ratio 24 0 0 7 18 2.57 48 0 13 7 24 1.26

As seen in Table 4, the mixtures of the present invention provided amore than additive effect. By using the following formula, Applicant wasable to determine that this response was synergistic: %C_(exp)=A+B−(AB/100).

Bacillus thuringiensis subsp. aizawai was applied at a concentration of10 ppm (10 μg/ml). Bacillus thuringiensis subsp. kurstaki was applied ata concentration of 3.125 ppm (3.125 μg/ml). The Bacillus thuringiensissubsp. aizawai/Bacillus thuringiensis subsp. kurstaki mixture wasapplied at a concentration of 10 ppm Bacillus thuringiensis subsp.aizawai and 3.125 ppm Bacillus thuringiensis subsp. kurstaki.

The results of this calculation indicated that the synergy ratio was2.57 at 24 hours and 1.26 at 48 hours. As a finding of higher than 1 isindicative of synergy, the ratios of 1.26 and above are synergistic.Synergy was shown at a ratio of Bacillus thuringiensis subsp. kurstakito Bacillus thuringiensis subsp. aizawai of 1:3.2.

Example 5 Southwestern Corn Borer

In this study, the response of southwestern corn borer larvae tosynergistic amounts of Bacillus thuringiensis subsp. aizawai andBacillus thuringiensis subsp. kurstaki was observed. The results of thisstudy can be seen below in Table 5.

TABLE 5 % Efficacy Time after Neg. treatment Control Btk + Bta Synergy(h) dH₂O Bta Btk (Ratio 1:3.2) Ratio 24 4 10 4 11 NS 48 4 14 18 18 NS

As seen in Table 5, the mixtures of the present invention provided amore than additive effect. By using the following formula, Applicant wasable to determine that this response was synergistic: %C_(exp)=A+B−(AB/100).

Bacillus thuringiensis subsp. aizawai was applied at a concentration of10 ppm (10 μg/ml). Bacillus thuringiensis subsp. kurstaki was applied ata concentration of 3.125 ppm (3.125 μg/ml). The Bacillus thuringiensissubsp. aizawai/Bacillus thuringiensis subsp. kurstaki mixture wasapplied at a concentration of 10 ppm Bacillus thuringiensis subsp.aizawai and 3.125 ppm Bacillus thuringiensis subsp. kurstaki.

The results of this calculation indicated that the there was no synergyat 24 and 48 hours.

Example 6 Soybean Looper

In this study, the response of soybean looper larvae to synergisticamounts of Bacillus thuringiensis subsp. aizawai and Bacillusthuringiensis subsp. kurstaki was observed. The results of this studycan be seen below in Table 6.

TABLE 6 % Efficacy Time after Neg. treatment Control Btk + Bta Synergy(h) dH₂O Bta Btk (Ratio 1:2.5) Ratio 24 0 7 4 21 1.96 48 0 14 8 41 1.96

As seen in Table 6, the mixtures of the present invention provided amore than additive effect. By using the following formula, Applicant wasable to determine that this response was synergistic: %C_(exp)=A+B−(AB/100).

Bacillus thuringiensis subsp. aizawai was applied at a concentration of25 ppm (25 μg/ml). Bacillus thuringiensis subsp. kurstaki was applied ata concentration of 10 ppm (10 μg/ml). The Bacillus thuringiensis subsp.aizawai/Bacillus thuringiensis subsp. kurstaki mixture was applied at aconcentration of 25 ppm Bacillus thuringiensis subsp. aizawai and 10 ppmBacillus thuringiensis subsp. kurstaki.

The results of this calculation indicated that the synergy ratio was1.96 at 24 and 48 hours. As a finding of higher than 1 is indicative ofsynergy, the ratio of 1.96 is synergistic. Synergy was shown at a ratioof Bacillus thuringiensis subsp. kurstaki to thuringiensis subsp.aizawai of 1:2.5.

Example 7 Corn Earworm

In this study, the response of corn earworm larvae to synergisticamounts of Bacillus thuringiensis subsp. aizawai and Bacillusthuringiensis subsp. kurstaki was observed. The results of this studycan be seen below in Table 7.

TABLE 7 % Efficacy Time after Neg. treatment Control Btk + Bta Synergy(h) dH₂O Bta Btk (Ratio 1:2.5) Ratio 24 0 7 7 27 2.0 48 0 20 20 36 1.0

As seen in Table 7, the mixtures of the present invention provided amore than additive effect. By using the following formula, Applicant wasable to determine that this response was synergistic: %C_(exp)=A+B−(AB/100).

Bacillus thuringiensis subsp. aizawai was applied at a concentration of25 ppm (25 μg/ml). Bacillus thuringiensis subsp. kurstaki was applied ata concentration of 10 ppm (10 μg/ml). The Bacillus thuringiensis subsp.aizawai/Bacillus thuringiensis subsp. kurstaki mixture was applied at aconcentration of 25 ppm Bacillus thuringiensis subsp. aizawai and 10 ppmBacillus thuringiensis subsp. kurstaki.

The results of this calculation indicated that the synergy ratio was 2.0at 24 hours. As a finding of higher than 1 is indicative of synergy, theratio of 2.0 is synergistic. Synergy was shown at a ratio of Bacillusthuringiensis subsp. kurstaki to Bacillus thuringiensis subsp. aizawaiof 1:2.5.

In summary, synergy was seen against diamondback moth, beet armyworm,sugarcane borer, soybean looper, corn earworm, cabbage looper andsouthwestern corn borer.

1. A method of controlling a crop plant pest selected from the groupconsisting of Diamondback moth (Plutella xylostella), Beet armyworm(Spodoptera exigua), Sugarcane borer (Diatraea saccharalis), Soybeanlooper (Chrysodeixis includens), Corn earworm (Helicoverpa zea), CabbageLooper (Trichoplusia ni), and Southwestern Corn Borer (Diatraeagrandiosella) comprising applying a synergistic amount of Bacillusthuringiensis subsp. aizawai and Bacillus thuringiensis subsp. kurstakito a plant, wherein the ratio of Bacillus thuringiensis subsp. kurstakito Bacillus thuringiensis subsp. aizawai is from about 1:0.001 to about1:3.5.
 2. The method of claim 1 wherein the ratio of Bacillusthuringiensis subsp. kurstaki to Bacillus thuringiensis subsp. aizawaiis from about 1:0.01 to about 1:3.
 3. The method of claim 1 wherein theratio of Bacillus thuringiensis subsp. kurstaki to Bacillusthuringiensis subsp. aizawai is from about 1:0.1 to about 1:2.5.
 4. Themethod of claim 1 wherein the amount of Bacillus thuringiensis subsp.kurstaki and Bacillus thuringiensis subsp. aizawai is from about 5 toabout 400 grams per hectare.
 5. The method of claim 1 wherein the amountof Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensissubsp. aizawai is from about 10 to about 350 grams per hectare.
 6. Themethod of claim 1 wherein the amount of Bacillus thuringiensis subsp.kurstaki and Bacillus thuringiensis subsp. aizawai is from about 25 toabout 300 grams per hectare.
 7. The method of claim 1 wherein the cropplant pest is Diamondback moth (Plutella xylostella).
 8. The method ofclaim 1 wherein the crop plant pest is Beet armyworm (Spodopteraexigua).
 9. The method of claim 1 wherein the crop plant pest isSugarcane borer (Diatraea saccharalis).
 10. The method of claim 1wherein the crop plant pest is Soybean looper (Chrysodeixis includens).11. The method of claim 1 wherein the crop plant pest is Corn earworm(Helicoverpa zea).
 12. The method of claim 1 wherein the crop plant pestis Cabbage Looper (Trichoplusia ni).
 13. The method of claim 1 whereinthe crop plant pest is Southwestern Corn Borer (Diatraea grandiosella).14. The method of claim 1 wherein the plant is selected from the groupconsisting of root and tuber vegetables, bulb vegetables, leafynon-brassica vegetables, leafy brassica vegetables, succulent or driedlegumes, fruiting vegetables, cucurbit vegetables, citrus fruits, pomefruits, stone fruits, berry and small fruits, tree nuts, cereal grains,forage and fodder grasses and hay, non-grass animal feeds, herbs,spices, flowers, bedding plants, ornamental flowers, artichoke,asparagus, coffee, cotton, tropical fruits, hops, malanga, peanut,pomegranate, oil seed vegetables, sugarcane, tobacco, turf, andwatercress.
 15. The method of claim 14 wherein the plant is geneticallymodified.
 16. The method of claim 15 wherein the cereal grains areselected from the group consisting of barley, buckwheat, pearl millet,proso millet, oats, corn, field corn, sweet corn, seed corn, popcorn,rice, rye, sorghum (milo), sorghum species, grain sorghum, sudangrass(seed), teosinte, triticale, wheat, wild rice, and cultivars, varietiesand hybrids thereof.
 17. The method of claim 16 wherein the plant isgenetically modified corn.
 18. The method of claim 14 wherein thesucculent or dried vegetable legumes are selected from the groupconsisting of Lupinus beans, Phaseolus beans, Vigna beans, broad beans,chickpea, guar, jackbean, lablab bean, lentil, Pisum peas, pigeon pea,soybean, immature seed soybean, sword bean, peanut, and cultivars,varieties and hybrids thereof.
 19. The method of claim 18 wherein theplant is genetically modified soybean.